Rotor-Gene Probe PCR Kit

For ultrafast, real-time PCR and two-step RT-PCR using sequence-specific probes

Sensitive detection of even low copy numbers

Accurate detection of a wide range of template amounts

Optimized for ultrafast, reliable results on Rotor-Gene cyclers

Specially formulated, ready-to-use master mix for fast cycling

The Rotor-Gene Probe PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly sensitive quantification of gDNA and cDNA targets with real-time PCR and two-step RT-PCR using sequence-specific probes. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

Real-time PCR was carried out using two-fold dilutions of human genomic DNA (30 ng to 3.75 ng) and a self-designed TaqMan assay for IL1R2 (interleukin 1 receptor, type II). Reactions were run using either [A] the Rotor-Gene Probe PCR Kit and Rotor-Gene Q or [B] a kit and cycler from Supplier AII (5 replicates per template dilution). The Rotor-Gene system provided lower CT values, greater reproducibility within replicates, and greater resolution of different template dilutions.|Real-time PCR analysis of human genomic DNA was carried out using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. [A] Analysis of two-fold dilutions of DNA (from 30 ng [5000 copies] to 0.06 ng [20 copies]) using a self-designed TaqMan assay for IL1R2 (interleukin 1 receptor, type II); 5 replicates per template dilution. The average difference in CT value between adjacent template dilutions was 1.07 cycles. [B] Analysis of 5 ng DNA using a self-designed TaqMan assay for IL1R2. From 100 replicates, the standard deviation was 0.21. [C] Analysis of 20 ng DNA using a self-designed TaqMan assay for ACTB (actin, beta). From 100 replicates, the standard deviation was 0.29.|Real-time PCR was carried out using fourfold dilutions of human genomic DNA (equivalent to 16, 666 cells down to 1 cell) and a self-designed TaqMan assay for ACTB (actin, beta). Reactions were run in triplicate using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. The Rotor-Gene system provided reliable detection over the entire range of template dilutions, with high reproducibility within each set of triplicates.|Real-time PCR was carried out using ten-fold dilutions of plasmid DNA (109 to 10 copies) and a self-designed TaqMan assay for PPIA (cyclophilin A). Reactions were run in triplicate using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. The Rotor-Gene system provided reliable detection over the entire range of template dilutions as well as highly reproducible CT values within each set of triplicates.|Cations in the Rotor-Gene Q PCR buffer increase specific primer annealing. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in Rotor-Gene Probe PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|

Real-time PCR was carried out using ten-fold dilutions of plasmid DNA (109 to 10 copies) and a self-designed TaqMan® assay for PPIA (cyclophilin A).

Principle

The Rotor-Gene Probe PCR Kit enables reliable real-time two-step RT-PCR quantification on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. The kit is designed for use with hydrolysis probes (e.g., TaqMan® probes). Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that enables cycler run times of as low as 45 minutes (see figure "Fast primer annealing").

Components of 2x Rotor-Gene Probe PCR Kit*

Component

Features

Benefits

HotStarTaq Plus DNA Polymerase

5 min activation at 95ºC

Set up of qPCR reactions at room temperature

Rotor-Gene Probe PCR Buffer

Balanced combination of NH4+ and K+ ions

Specific primer annealing ensures reliable qPCR results

Unique Q-Bond additive

Faster PCR run times enable faster results and more reactions per day

* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).

Procedure

The ready-to-use Rotor-Gene Probe PCR Master Mix eliminates the need for optimization of reaction and cycling conditions. Just add template DNA, primers, and probe to the master mix and program the cycler. Instructions are provided in the detailed handbook supplied with the kit.

Hydrolysis probes (e.g., TaqMan® probes) can be used in combination with the Rotor-Gene Probe PCR Kit on the Rotor-Gene Q for fast and sensitive quantification — simply add the primer-probe mix and template to the master mix.

The Rotor-Gene Probe PCR Kit is for fast, real-time PCR and two-step RT-PCR of gDNA or cDNA targets using sequence-specific probes on the Rotor-Gene Q. It is also compatible with Rotor-Gene 3000 and the Rotor-Gene 6000 cyclers.